HIV Diagnosis and Monitoring of Antiretroviral Therapy
Re
se
vi
d
r
Ve
on
si
20
09
Laboratory support is critical in all the areas of HIV diagnosis and
management. Diagnosis of HIV infection cannot be established by
any means other than blood tests by the laboratory. CD4 lymphocyte
count is a prerequisite for the initiation of antiretroviral therapy and
for monitoring treatment outcome. Both immunological and
microbiological monitoring of antiretroviral therapy is therefore
exclusively dependent on an efficient laboratory service. While
laboratory support to HIV/AIDS programmes is very important, the
infrastructure, expertise and networking require strengthening in
most countries of our Region. These Guidelines aim to assist Member
Countries in scaling up ART and responding to the rapidly evolving
HIV/AIDS epidemic.
World Health House
Indraprastha Estate,
Mahatma Gandhi Marg,
New Delhi-110002, India
Website: www.searo.who.int
SEA-HLM-382
Guidelines for
HIV Diagnosis
and Monitoring of
Antiretroviral Therapy
SEA-HLM-382 (Rev. 2)
Distribution: General
Revised Version 2009
Guidelines for
HIV Diagnosis and Monitoring
of Antiretroviral Therapy
© World Health Organization 2009
All rights reserved.
Requests for publications, or for permission to reproduce or translate WHO
publications – whether for sale or for noncommercial distribution – can be
obtained from Publishing and Sales, World Health Organization, Regional
Office for South-East Asia, Indraprastha Estate, Mahatma Gandhi Marg, New
Delhi 110 002, India (fax: +91 11 23370197; e-mail: publications@searo.
who.int).
The designations employed and the presentation of the material in this
publication do not imply the expression of any opinion whatsoever on the part
of the World Health Organization concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its
frontiers or boundaries. Dotted lines on maps represent approximate border
lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products
does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that are not
mentioned. Errors and omissions excepted, the names of proprietary products
are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization
to verify the information contained in this publication. However, the published
material is being distributed without warranty of any kind, either expressed or
implied. The responsibility for the interpretation and use of the material lies
with the reader. In no event shall the World Health Organization be liable for
damages arising from its use.
This publication does not necessarily represent the decisions or policies of the
World Health Organization.
Printed in India
Contents
Acronyms and abbreviations .......................................................................v
Preface to First Edition ............................................................................... vii
1.
HIV and laboratories: An overview.................................................... 1
2.
Diagnosis of HIV infection................................................................. 7
3.
Immunological monitoring of antiretroviral therapy: CD4 T
lymphocytes counts ........................................................................ 17
4.
Virological monitoring of ART ......................................................... 27
5.
Laboratory monitoring of side-effects of ART ................................... 35
6.
Tuberculosis in HIV/AIDS ................................................................ 39
7.
New technologies in HIV diagnosis and ART monitoring ................. 47
8.
Laboratory infrastructure and networking ........................................ 53
9.
Quality system in the laboratory ..................................................... 57
10.
Bio-safety practices, accidental exposures and post-exposure
prophylaxis .................................................................................... 61
11.
Collection, storage, packaging and transport of
biological specimens ....................................................................... 71
Suggested further reading ......................................................................... 81
Annexes
1.
Suggested sources of availability of laboratory equipment
and supplies ................................................................................... 85
2.
Collection and storage of clinical specimens .................................... 87
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
iii
3.
Procedures carrying potential risks of HIV, HBV and
other bloodborne agents ................................................................. 89
4.
Summary of CD4 T lymphocytes enumeration technologies:
flow cytometry ................................................................................ 91
5.
Summary of CD4 T lymphocytes enumeration technologies:
dedicated and manual assays .......................................................... 93
6.
Summary of main characteristics of viral load technologies
based on Nucleic Acid Testing (NAT) ............................................... 95
7.
Summary of main characteristics of viral load technologies
not based on nucleic acid (non-NAT) .............................................. 97
8.
Good laboratory practices ............................................................... 99
9.
Algorithm for determination of infection status in
HIV-exposed children 98%). For infants born to HIV-1–infected mothers, it has been recommended
that diagnostic testing with HIV-1 DNA or RNA assays be performed within the
first 14 days of life, at one to two months of age, and again at three to six months
of age. If any of these test results are positive, repeat testing is recommended to
confirm the diagnosis of HIV-1 infection. However, if an infant is breastfeeding
he or she remains at risk of acquiring HIV infection throughout the breastfeeding
period and, therefore, negative virological test results can be assumed to reliably
indicate HIV infection status only after six weeks after complete cessation of
breastfeeding (Annex 9).
Alternatively, if the first PCR is negative in a non-breastfed baby, confirm
with a second PCR test at six months. Definitive exclusion of HIV-1 infection is
based on two negative virological test results, one obtained at one month of age
and one obtained at four months of age, or two negative HIV-1 antibody test
results from separate specimens obtained at six months of age. For exclusion
of infection, the child should have no other laboratory or clinical evidence of
HIV-1 infection and should not be breastfed.
Dried blood spot (DBS) represents a paradigm shift in accessibility to
nucleic acid testing for HIV infection. Nucleic acids in DBS have been shown
to be stable for several months at ambient temperatures, provided the DBS
specimens have been thoroughly dried and stored with desiccant. Thus DBS
specimens can be collected at remote rural sites and transported to a central
or regional testing laboratory.
10
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
However, given the expense and complexity of nucleic acid testing,
the World Health Organization strongly encourages the development of
technologically simpler, less expensive assays that can be used to diagnose
HIV-1 infection in early infancy. The ultra-sensitive p24 (Up24 assay) assay is
gaining support as a tool for detection of HIV-1 infection in infants following
mother-to-child transmission. The ultrasensitive p24 antigen assay is an ELISA
and is, therefore, suited to health facilities where serologic al testing is routinely
performed. The performance of the Up24 assay for HIV diagnosis in infants
and young children has been evaluated in a number of studies in countries
with different HIV subtypes yielding sensitivities and specificities ranging from
96% to 99% when compared to HIV DNA PCR testing.
By the age of 12 months most uninfected HIV-exposed children lose
maternal antibodies and testing HIV antibody-positive at this age can be
considered indicative of HIV infection. This should be confirmed by repeat
antibody testing after the age of 18 months. If an HIV-exposed child between 9
and 18 months of age had never been breastfed or had stopped breastfeeding
for at least six weeks and had a negative HIV antibody test result, then the
child should be considered uninfected with no further testing required unless
symptoms develop.
Specimen collection
Optimal time of specimen
collection
Blood specimens can be collected at any
convenient time
Correct specimen type and
method of collection
Whole blood or anticoagulated blood
Adequate quantity
Approximately 3−5 ml
DBS may also be collected. However, it is
advisable to collect multiple spots for repeat assay
or quality control
Specimen transport and storage
Time between
specimen collection
and processing
• Whole blood specimens should be transported
at ambient temperature (20 °C to 25 °C) and
processed as soon as possible or within 24 hours.
• If serum/plasma has been separated, it can be
stored in a refrigerator (2 °C to 8 °C) for a week or
can be transported at 2 °C to 8 °C
• For longer storage, store in a freezer at –20 °C
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
11
Criteria for rejection of sample
The following are the criteria for the sample rejection:
•
Hemolyzed samples
•
Samples showing turbidity
•
Specimens not stored and transported properly
•
Sample that does not carry appropriate label
•
Samples that have leaked
Table 2.2: UNAIDS and WHO recommendations for HIV testing
strategies according to test objective and prevalence of
infection in the sample population
Objective of testing
Prevalence of infection in the
category to which the patient belongs
(percentage)
Testing
strategy
applicable
−
I
>10
30
10
2 times) indeterminate results.
Reporting procedure
Report
Negative – if initial/screening test shows non-reactive result.
Positive – if the sample shows reactive results concordantly by the
three tests as per the algorithm.
HIV-1 positive/HIV-2 positive or dual reactive as per the algorithm
should be mentioned.
In case of dual reactivity, confirmation by molecular assays may
be considered.
Indeterminate – if the sample shows discordant results by the
three tests. The follow-up testing is recommended as mentioned
above.
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
13
Selection of test kits
A wide variety of test kits are now available for HIV diagnosis. The selection
of appropriate test kits/assays/reagents is critical to ensure quality in laboratory
services. Every country or laboratory must, therefore, define a policy for
selection, evaluation and procurement of the most appropriate kit. Selection
of assays/reagents is a complex process that needs to be planned carefully. The
overall performance of an assay/reagent depends upon a number of local factors.
Quite often the manufacturer’s quoted sensitivity and specificity figures may
not reflect the actual working figures. Therefore, selection of an assay/reagent
needs to consider the testing needs of a centre and the resources available to
meet those needs. Procurement systems may have a significant impact on the
selection of kits. Stock control is vital, especially where continuity of supply
cannot be guaranteed. Ongoing monitoring systems are essential to identify
problems either with the assay/reagent or the laboratory.
The steps that are involved in the selection of a test kit/reagent for a
testing laboratory are:
14
(1)
Sensitivity and specificity of the test: Sensitivity is the ability of
an assay to identify all infected individuals (true positives). The
specificity of an assay is the ability of an assay to identify uninfected
individuals (true negatives). Kits/assays should have a sensitivity
>99% for Rapid test/ELISA) and specificity >98%.
(2)
Ease of testing, competency of the staff performing the test and
infrastructure available.
(3)
Type of sample: serum/plasma/whole blood/dried blood spot/other
body fluid
(4)
Type of test controls provided: Reliable kit systems that provide
internal controls are preferred. Similarly, for rapid tests, kits that
provide a sample addition check are generally preferred.
(5)
Number of tests per kit.
(6)
Shelf life of kit as per the kit recommendation: Kits with a longer
expiry date are preferred over those that have more recent expiry
dates.
(7)
Storage and handling requirements during transport and their
feasibility.
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
(8)
Resource availability: Finally, the choice of a test kit/reagent would
depend a great deal on the availability of financial resources, existing
systems in place in the laboratory and the time scale in which results
are expected to be delivered.
(9)
Time required to complete the test: If HIV results are required to
be obtained within a short time and only a few samples need to be
tested in a laboratory, rapid and simple HIV kits may be preferred
over other assays. On the contrary, if a large number of samples are
to be tested, assays such as ELISA may be preferred.
Evaluation and final selection of kit
Define specific requirements for assays/reagents for the country
Prepare a protocol for laboratory assessment
Collect all available relevant data pertaining to kits/reagents
Assess on paper each assay/reagent against specific requirements and list most
suitable assays
Perform laboratory assessment of most suitable candidate kits
Evaluate results
Select assay/reagent
Evaluation and procurement
Procurement of test kits/reagents depends on several factors which need to be
considered. These factors in turn would depend on the logistics and practices
that are prevalent in the laboratory, such as mechanism of procurement of kits,
specific requirements of laboratory and time-frame for procurement.
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
15
Monitoring subsequent performance
Monitoring the performance of a reagent/kit is a continuous process which
begins from the time of procurement until all the kits are used or reach their
expiry dates. Each country should draw up a plan for periodic monitoring of
reagents/kits at various testing levels in the country, much akin to those that
are already existing for vaccines in many countries. It may be advisable to have
periodic “post-marketing surveillance” of the kits carried out in the central
laboratory which gets these kits for evaluation from various peripheral testing
laboratories.
Technical support from WHO
Realizing the inadequacies in the mechanisms at the country level to procure
reagents/kits, WHO has established a mechanism called AIDS Medicines and
Diagnostic Services (AMDS) with the following features:
•
AMDS is created to expand access to quality, effective anti-retroviral
therapy (ART) by facilitating increased supply of drugs and diagnostics
in developing countries.
•
AMDS will provide to manufacturers information and forecasting
about global demand/market.
•
AMDS will provide to buyers sources, process and patents on drugs
and diagnostics and assist them in obtaining the best prices for
individual or pooled demand.
•
AMDS will provide technical support to countries in improving their
procurement mechanisms.
•
AMDS will assist countries, NGOs and other non-profit
organizations.
AMDS has also created a pre-qualification project (PQP) which aims at
ensuring quality, safety and efficacy of HIV/AIDS medicines and diagnostics.
It assesses products voluntarily submitted by manufacturers and certifies their
conformation to WHO standards. The approved drugs and diagnostics are
shown in the public domain. Twenty-three HIV kits are available at present.
CD4 and viral load kits are also being monitored. These services are managed
by WHO in collaboration with UNICEF, UNAIDS, UNFPA and the World Bank.
AMDS, however, will not provide free drugs or diagnostics to the countries. All
the information pertaining to AMDS can be accessed from the WHO website
(www.who.int).
16
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
3
Immunological monitoring of
antiretroviral therapy:
CD4 T lymphocytes counts
What are CD4 T lymphocytes?
Cellular components of blood comprise red blood cells and white blood cells.
Two populations of leucocytes constitute the latter — the granulocytes and nongranulocytes, including the lymphocytes. Surface receptors of the lymphocytes
provide identity to sub-populations of lymphocytes which differentiate into
unique clusters. This property gives the subtypes of lymphocytes a nomenclature
of clusters of differentiation followed by the number of the unique subtype
(CD1, CD2, CD3, CD4…). CD stands for cluster of differentiation; CD numbers
are now used to identify cell surface antigens that can be distinguished by
monoclonal antibodies. CD4 T lymphocytes (CD4+ T-cells), commonly known
as T helper cells, play a vital role in maintaining the integrity of the human
immune system.
Importance of CD4 T lymphocytes
A primary target of HIV is CD4 T lymphocytes which are preferentially depleted
during the course of the disease. It is well recognized now that accurate and
reliable enumeration of CD4 T lymphocytes is very crucial for monitoring the
rate of progression to AIDS, both for initiating prophylaxis for opportunistic
infections as well as monitoring the impact of antiretroviral therapy (ART).
Methods of enumeration of CD4 T lymphocytes
Immunofluorescence analysis by flow cytometry (FCM) is the gold standard for
CD4 T lymphocytes measurements. FCM is the first choice, if large number of
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
17
specimens need to be tested. To obtain an absolute CD4 T lymphocytes count,
two concepts (Annex 4 and 5) are utilized:
Dual-platform (DP) approach
The DP approach uses two instruments to generate absolute CD4 T
lymphocytes: an FCM for generating a percentage CD4 T lymphocytes among
lymphocytes and a haematological analyser to enumerate the absolute
lymphocyte counts. An absolute CD4 T-cell count is derived by multiplying %
CD4 T lymphocytes by the absolute lymphocyte count. Examples of these DP
FCM instruments include BD FACSVantage, FACSCalibur/FASCScan/FACSort
or Beckman-Coulter Epics XL/XL-MCL.
Single-platform (SP) approach
This technique enables absolute CD4 T lymphocytes counts to be derived
directly without the need for a haematological analyzer, i.e., the use of volumetric
counting (Partec CyFlow), microfluorometry (Guava) and, most commonly, the
addition of reference fluorescent beads of a known density (eg., BD-Truecount
and Coulter Flowcount) to the sample.
Other alternative methods
Flow cytometry is a widely used method for estimation of CD4 T lymphocytes.
Flow cytometer and reagents are expensive and hence are a cause for concern
in developing countries. For those countries and settings where the infrastructure
is not available or difficult to set up for such FCM technologies, a number of
alternative assays have been developed and most of them are commercially
available. These assays have been evaluated against the gold standard method
and reported in literature.
Total lymphocyte count
In cases where CD4 testing cannot be assessed, the presence of a total
lymphocyte count (TLC) of 1200 cells/ul or below may be used as a substitute
indication for ARV treatment in symptomatic HIV-infected patients. While the
total lymphocyte count correlates poorly with the CD4 T lymphocytes count
in asymptomatic patients, in combination with clinical staging it is still a useful
marker of prognosis and survival.
18
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
There are limitations in the use of TLC as a substitute for CD4 T
lymphocytes counts. For example:
•
Lack of correlation with CD4 counts in asymptomatic patients
•
Variation due to intercurrent infections or drugs.
Microscope-based CD4 counting systems
These alternative assays are, however, fairly labour intensive and thus less
appropriate for a large number of samples. Moreover, qualified personnel are
required for making accurate measurements.
•
Microbead system (Dynal Biotech, Oslo, Norway) uses two types
of beads. The first bead removes monocytes from the sample and
the second (CD4) estimates CD4 T lymphocytes that get stained
with acridine orange to make the cell nuclei visible for counting
under a fluorescent microscope. The initial cost of equipment for a
fluorescence microscope is low (US$ 6000) with a running cost of
US$ 3–5 per test. A modified DynaBeads system with an alternate
stain for the cells can be used with a light microscope.
Evaluations of Dynal bead assay was highly comparable with the standard
FCM and found to be accurate and reproducible. However, this is a manual
and labour-intensive assay. To scale these up to match expanding access to ART
may prove a challenge. The system could be cheaper than other alternatives
and will be useful in small settings if it is backed up with flow cytometry for
quality assurance.
Affordable flow cytometry
Combined use of CD4 and CD45 conjugated antibodies using Panleucogating
(PLG) methodology (eg. Beckman Coulter FlowCARE CD4 Reagent) has been
found to be workable and cost-effective.
Modified single platform volumetric flow cytometry
Cyflow from Partec
The system works on volumetric method, i.e. a known amount of blood with
a single antibody reagent. It can also be run on solar panels and car batteries,
and hence may be used in remote areas. The methodology is simple to carry
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
19
out based on an SP method with single antibody reagent and ten-minute
incubation. The system showed good correlation with the CD4 T lymphocytes
counts obtained by conventional flow cytometry. However, an experienced
technician is required for accurate measurement. The CyFlow capital equipment
costs approximately US$ 20 000 and the cost per test is US$2.
Guava technology
A SP system that uses CD3 antibodies to measure T lymphocytes and CD4
antibodies to estimate absolute T-cells expressing CD4. The system showed
good correlation with conventional flow cytometry and is easy to operate. It
uses smaller blood volume. It requires very minimal infrastructure facilities and
it is easy to train technologists to perform the test. The cost per test is around
US$ 2. However, the equipment costs about US$ 26 000.
The recent evaluation studies with these two modified flowcytometry
systems have shown a good correlation with the standard FCM.
Selection of alternative methodology for CD4 T
lymphocytes count
The following specifications should be considered for the selection of the better
technology for CD4 T lymphocytes count:
•
The equipment should be simple to operate, easy to maintain and
require minimal training.
•
Methodology needs minimal infrastructure laboratory facilities.
•
Methodology should have the internal QC procedures.
•
Test kits should be cost-effective and available anytime.
•
Easy access to the technical specialist/service engineer support.
•
Supplying company should be in a position to supply the critical parts
of the equipment with short turnaround time.
For HIV/ART monitoring, all CD4 T lymphocytes estimation for the same
patient must be done using the same technology and same laboratory to
ensure comparability.
20
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
Utility of CD4 T lymphocytes count in monitoring
According to the WHO recommendation, HIV-infected adults should start ARV
therapy when infection has been confirmed and one of the following conditions
is present (Table 3.1):
Table 3.1: WHO recommendations for initiating ARV therapy in
HIV-infected adults and adolescents according to
CD4 T lymphocytes counts and total lymphocyte counts*
WHO Stage
CD4 T lymphocytes counts
Total lymphocyte counts
IV
Irrespective
Irrespective
III
se
vi
d
r
Ve
on
si
20
09
Laboratory support is critical in all the areas of HIV diagnosis and
management. Diagnosis of HIV infection cannot be established by
any means other than blood tests by the laboratory. CD4 lymphocyte
count is a prerequisite for the initiation of antiretroviral therapy and
for monitoring treatment outcome. Both immunological and
microbiological monitoring of antiretroviral therapy is therefore
exclusively dependent on an efficient laboratory service. While
laboratory support to HIV/AIDS programmes is very important, the
infrastructure, expertise and networking require strengthening in
most countries of our Region. These Guidelines aim to assist Member
Countries in scaling up ART and responding to the rapidly evolving
HIV/AIDS epidemic.
World Health House
Indraprastha Estate,
Mahatma Gandhi Marg,
New Delhi-110002, India
Website: www.searo.who.int
SEA-HLM-382
Guidelines for
HIV Diagnosis
and Monitoring of
Antiretroviral Therapy
SEA-HLM-382 (Rev. 2)
Distribution: General
Revised Version 2009
Guidelines for
HIV Diagnosis and Monitoring
of Antiretroviral Therapy
© World Health Organization 2009
All rights reserved.
Requests for publications, or for permission to reproduce or translate WHO
publications – whether for sale or for noncommercial distribution – can be
obtained from Publishing and Sales, World Health Organization, Regional
Office for South-East Asia, Indraprastha Estate, Mahatma Gandhi Marg, New
Delhi 110 002, India (fax: +91 11 23370197; e-mail: publications@searo.
who.int).
The designations employed and the presentation of the material in this
publication do not imply the expression of any opinion whatsoever on the part
of the World Health Organization concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its
frontiers or boundaries. Dotted lines on maps represent approximate border
lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products
does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that are not
mentioned. Errors and omissions excepted, the names of proprietary products
are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization
to verify the information contained in this publication. However, the published
material is being distributed without warranty of any kind, either expressed or
implied. The responsibility for the interpretation and use of the material lies
with the reader. In no event shall the World Health Organization be liable for
damages arising from its use.
This publication does not necessarily represent the decisions or policies of the
World Health Organization.
Printed in India
Contents
Acronyms and abbreviations .......................................................................v
Preface to First Edition ............................................................................... vii
1.
HIV and laboratories: An overview.................................................... 1
2.
Diagnosis of HIV infection................................................................. 7
3.
Immunological monitoring of antiretroviral therapy: CD4 T
lymphocytes counts ........................................................................ 17
4.
Virological monitoring of ART ......................................................... 27
5.
Laboratory monitoring of side-effects of ART ................................... 35
6.
Tuberculosis in HIV/AIDS ................................................................ 39
7.
New technologies in HIV diagnosis and ART monitoring ................. 47
8.
Laboratory infrastructure and networking ........................................ 53
9.
Quality system in the laboratory ..................................................... 57
10.
Bio-safety practices, accidental exposures and post-exposure
prophylaxis .................................................................................... 61
11.
Collection, storage, packaging and transport of
biological specimens ....................................................................... 71
Suggested further reading ......................................................................... 81
Annexes
1.
Suggested sources of availability of laboratory equipment
and supplies ................................................................................... 85
2.
Collection and storage of clinical specimens .................................... 87
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
iii
3.
Procedures carrying potential risks of HIV, HBV and
other bloodborne agents ................................................................. 89
4.
Summary of CD4 T lymphocytes enumeration technologies:
flow cytometry ................................................................................ 91
5.
Summary of CD4 T lymphocytes enumeration technologies:
dedicated and manual assays .......................................................... 93
6.
Summary of main characteristics of viral load technologies
based on Nucleic Acid Testing (NAT) ............................................... 95
7.
Summary of main characteristics of viral load technologies
not based on nucleic acid (non-NAT) .............................................. 97
8.
Good laboratory practices ............................................................... 99
9.
Algorithm for determination of infection status in
HIV-exposed children 98%). For infants born to HIV-1–infected mothers, it has been recommended
that diagnostic testing with HIV-1 DNA or RNA assays be performed within the
first 14 days of life, at one to two months of age, and again at three to six months
of age. If any of these test results are positive, repeat testing is recommended to
confirm the diagnosis of HIV-1 infection. However, if an infant is breastfeeding
he or she remains at risk of acquiring HIV infection throughout the breastfeeding
period and, therefore, negative virological test results can be assumed to reliably
indicate HIV infection status only after six weeks after complete cessation of
breastfeeding (Annex 9).
Alternatively, if the first PCR is negative in a non-breastfed baby, confirm
with a second PCR test at six months. Definitive exclusion of HIV-1 infection is
based on two negative virological test results, one obtained at one month of age
and one obtained at four months of age, or two negative HIV-1 antibody test
results from separate specimens obtained at six months of age. For exclusion
of infection, the child should have no other laboratory or clinical evidence of
HIV-1 infection and should not be breastfed.
Dried blood spot (DBS) represents a paradigm shift in accessibility to
nucleic acid testing for HIV infection. Nucleic acids in DBS have been shown
to be stable for several months at ambient temperatures, provided the DBS
specimens have been thoroughly dried and stored with desiccant. Thus DBS
specimens can be collected at remote rural sites and transported to a central
or regional testing laboratory.
10
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
However, given the expense and complexity of nucleic acid testing,
the World Health Organization strongly encourages the development of
technologically simpler, less expensive assays that can be used to diagnose
HIV-1 infection in early infancy. The ultra-sensitive p24 (Up24 assay) assay is
gaining support as a tool for detection of HIV-1 infection in infants following
mother-to-child transmission. The ultrasensitive p24 antigen assay is an ELISA
and is, therefore, suited to health facilities where serologic al testing is routinely
performed. The performance of the Up24 assay for HIV diagnosis in infants
and young children has been evaluated in a number of studies in countries
with different HIV subtypes yielding sensitivities and specificities ranging from
96% to 99% when compared to HIV DNA PCR testing.
By the age of 12 months most uninfected HIV-exposed children lose
maternal antibodies and testing HIV antibody-positive at this age can be
considered indicative of HIV infection. This should be confirmed by repeat
antibody testing after the age of 18 months. If an HIV-exposed child between 9
and 18 months of age had never been breastfed or had stopped breastfeeding
for at least six weeks and had a negative HIV antibody test result, then the
child should be considered uninfected with no further testing required unless
symptoms develop.
Specimen collection
Optimal time of specimen
collection
Blood specimens can be collected at any
convenient time
Correct specimen type and
method of collection
Whole blood or anticoagulated blood
Adequate quantity
Approximately 3−5 ml
DBS may also be collected. However, it is
advisable to collect multiple spots for repeat assay
or quality control
Specimen transport and storage
Time between
specimen collection
and processing
• Whole blood specimens should be transported
at ambient temperature (20 °C to 25 °C) and
processed as soon as possible or within 24 hours.
• If serum/plasma has been separated, it can be
stored in a refrigerator (2 °C to 8 °C) for a week or
can be transported at 2 °C to 8 °C
• For longer storage, store in a freezer at –20 °C
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
11
Criteria for rejection of sample
The following are the criteria for the sample rejection:
•
Hemolyzed samples
•
Samples showing turbidity
•
Specimens not stored and transported properly
•
Sample that does not carry appropriate label
•
Samples that have leaked
Table 2.2: UNAIDS and WHO recommendations for HIV testing
strategies according to test objective and prevalence of
infection in the sample population
Objective of testing
Prevalence of infection in the
category to which the patient belongs
(percentage)
Testing
strategy
applicable
−
I
>10
30
10
2 times) indeterminate results.
Reporting procedure
Report
Negative – if initial/screening test shows non-reactive result.
Positive – if the sample shows reactive results concordantly by the
three tests as per the algorithm.
HIV-1 positive/HIV-2 positive or dual reactive as per the algorithm
should be mentioned.
In case of dual reactivity, confirmation by molecular assays may
be considered.
Indeterminate – if the sample shows discordant results by the
three tests. The follow-up testing is recommended as mentioned
above.
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
13
Selection of test kits
A wide variety of test kits are now available for HIV diagnosis. The selection
of appropriate test kits/assays/reagents is critical to ensure quality in laboratory
services. Every country or laboratory must, therefore, define a policy for
selection, evaluation and procurement of the most appropriate kit. Selection
of assays/reagents is a complex process that needs to be planned carefully. The
overall performance of an assay/reagent depends upon a number of local factors.
Quite often the manufacturer’s quoted sensitivity and specificity figures may
not reflect the actual working figures. Therefore, selection of an assay/reagent
needs to consider the testing needs of a centre and the resources available to
meet those needs. Procurement systems may have a significant impact on the
selection of kits. Stock control is vital, especially where continuity of supply
cannot be guaranteed. Ongoing monitoring systems are essential to identify
problems either with the assay/reagent or the laboratory.
The steps that are involved in the selection of a test kit/reagent for a
testing laboratory are:
14
(1)
Sensitivity and specificity of the test: Sensitivity is the ability of
an assay to identify all infected individuals (true positives). The
specificity of an assay is the ability of an assay to identify uninfected
individuals (true negatives). Kits/assays should have a sensitivity
>99% for Rapid test/ELISA) and specificity >98%.
(2)
Ease of testing, competency of the staff performing the test and
infrastructure available.
(3)
Type of sample: serum/plasma/whole blood/dried blood spot/other
body fluid
(4)
Type of test controls provided: Reliable kit systems that provide
internal controls are preferred. Similarly, for rapid tests, kits that
provide a sample addition check are generally preferred.
(5)
Number of tests per kit.
(6)
Shelf life of kit as per the kit recommendation: Kits with a longer
expiry date are preferred over those that have more recent expiry
dates.
(7)
Storage and handling requirements during transport and their
feasibility.
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
(8)
Resource availability: Finally, the choice of a test kit/reagent would
depend a great deal on the availability of financial resources, existing
systems in place in the laboratory and the time scale in which results
are expected to be delivered.
(9)
Time required to complete the test: If HIV results are required to
be obtained within a short time and only a few samples need to be
tested in a laboratory, rapid and simple HIV kits may be preferred
over other assays. On the contrary, if a large number of samples are
to be tested, assays such as ELISA may be preferred.
Evaluation and final selection of kit
Define specific requirements for assays/reagents for the country
Prepare a protocol for laboratory assessment
Collect all available relevant data pertaining to kits/reagents
Assess on paper each assay/reagent against specific requirements and list most
suitable assays
Perform laboratory assessment of most suitable candidate kits
Evaluate results
Select assay/reagent
Evaluation and procurement
Procurement of test kits/reagents depends on several factors which need to be
considered. These factors in turn would depend on the logistics and practices
that are prevalent in the laboratory, such as mechanism of procurement of kits,
specific requirements of laboratory and time-frame for procurement.
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
15
Monitoring subsequent performance
Monitoring the performance of a reagent/kit is a continuous process which
begins from the time of procurement until all the kits are used or reach their
expiry dates. Each country should draw up a plan for periodic monitoring of
reagents/kits at various testing levels in the country, much akin to those that
are already existing for vaccines in many countries. It may be advisable to have
periodic “post-marketing surveillance” of the kits carried out in the central
laboratory which gets these kits for evaluation from various peripheral testing
laboratories.
Technical support from WHO
Realizing the inadequacies in the mechanisms at the country level to procure
reagents/kits, WHO has established a mechanism called AIDS Medicines and
Diagnostic Services (AMDS) with the following features:
•
AMDS is created to expand access to quality, effective anti-retroviral
therapy (ART) by facilitating increased supply of drugs and diagnostics
in developing countries.
•
AMDS will provide to manufacturers information and forecasting
about global demand/market.
•
AMDS will provide to buyers sources, process and patents on drugs
and diagnostics and assist them in obtaining the best prices for
individual or pooled demand.
•
AMDS will provide technical support to countries in improving their
procurement mechanisms.
•
AMDS will assist countries, NGOs and other non-profit
organizations.
AMDS has also created a pre-qualification project (PQP) which aims at
ensuring quality, safety and efficacy of HIV/AIDS medicines and diagnostics.
It assesses products voluntarily submitted by manufacturers and certifies their
conformation to WHO standards. The approved drugs and diagnostics are
shown in the public domain. Twenty-three HIV kits are available at present.
CD4 and viral load kits are also being monitored. These services are managed
by WHO in collaboration with UNICEF, UNAIDS, UNFPA and the World Bank.
AMDS, however, will not provide free drugs or diagnostics to the countries. All
the information pertaining to AMDS can be accessed from the WHO website
(www.who.int).
16
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
3
Immunological monitoring of
antiretroviral therapy:
CD4 T lymphocytes counts
What are CD4 T lymphocytes?
Cellular components of blood comprise red blood cells and white blood cells.
Two populations of leucocytes constitute the latter — the granulocytes and nongranulocytes, including the lymphocytes. Surface receptors of the lymphocytes
provide identity to sub-populations of lymphocytes which differentiate into
unique clusters. This property gives the subtypes of lymphocytes a nomenclature
of clusters of differentiation followed by the number of the unique subtype
(CD1, CD2, CD3, CD4…). CD stands for cluster of differentiation; CD numbers
are now used to identify cell surface antigens that can be distinguished by
monoclonal antibodies. CD4 T lymphocytes (CD4+ T-cells), commonly known
as T helper cells, play a vital role in maintaining the integrity of the human
immune system.
Importance of CD4 T lymphocytes
A primary target of HIV is CD4 T lymphocytes which are preferentially depleted
during the course of the disease. It is well recognized now that accurate and
reliable enumeration of CD4 T lymphocytes is very crucial for monitoring the
rate of progression to AIDS, both for initiating prophylaxis for opportunistic
infections as well as monitoring the impact of antiretroviral therapy (ART).
Methods of enumeration of CD4 T lymphocytes
Immunofluorescence analysis by flow cytometry (FCM) is the gold standard for
CD4 T lymphocytes measurements. FCM is the first choice, if large number of
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
17
specimens need to be tested. To obtain an absolute CD4 T lymphocytes count,
two concepts (Annex 4 and 5) are utilized:
Dual-platform (DP) approach
The DP approach uses two instruments to generate absolute CD4 T
lymphocytes: an FCM for generating a percentage CD4 T lymphocytes among
lymphocytes and a haematological analyser to enumerate the absolute
lymphocyte counts. An absolute CD4 T-cell count is derived by multiplying %
CD4 T lymphocytes by the absolute lymphocyte count. Examples of these DP
FCM instruments include BD FACSVantage, FACSCalibur/FASCScan/FACSort
or Beckman-Coulter Epics XL/XL-MCL.
Single-platform (SP) approach
This technique enables absolute CD4 T lymphocytes counts to be derived
directly without the need for a haematological analyzer, i.e., the use of volumetric
counting (Partec CyFlow), microfluorometry (Guava) and, most commonly, the
addition of reference fluorescent beads of a known density (eg., BD-Truecount
and Coulter Flowcount) to the sample.
Other alternative methods
Flow cytometry is a widely used method for estimation of CD4 T lymphocytes.
Flow cytometer and reagents are expensive and hence are a cause for concern
in developing countries. For those countries and settings where the infrastructure
is not available or difficult to set up for such FCM technologies, a number of
alternative assays have been developed and most of them are commercially
available. These assays have been evaluated against the gold standard method
and reported in literature.
Total lymphocyte count
In cases where CD4 testing cannot be assessed, the presence of a total
lymphocyte count (TLC) of 1200 cells/ul or below may be used as a substitute
indication for ARV treatment in symptomatic HIV-infected patients. While the
total lymphocyte count correlates poorly with the CD4 T lymphocytes count
in asymptomatic patients, in combination with clinical staging it is still a useful
marker of prognosis and survival.
18
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
There are limitations in the use of TLC as a substitute for CD4 T
lymphocytes counts. For example:
•
Lack of correlation with CD4 counts in asymptomatic patients
•
Variation due to intercurrent infections or drugs.
Microscope-based CD4 counting systems
These alternative assays are, however, fairly labour intensive and thus less
appropriate for a large number of samples. Moreover, qualified personnel are
required for making accurate measurements.
•
Microbead system (Dynal Biotech, Oslo, Norway) uses two types
of beads. The first bead removes monocytes from the sample and
the second (CD4) estimates CD4 T lymphocytes that get stained
with acridine orange to make the cell nuclei visible for counting
under a fluorescent microscope. The initial cost of equipment for a
fluorescence microscope is low (US$ 6000) with a running cost of
US$ 3–5 per test. A modified DynaBeads system with an alternate
stain for the cells can be used with a light microscope.
Evaluations of Dynal bead assay was highly comparable with the standard
FCM and found to be accurate and reproducible. However, this is a manual
and labour-intensive assay. To scale these up to match expanding access to ART
may prove a challenge. The system could be cheaper than other alternatives
and will be useful in small settings if it is backed up with flow cytometry for
quality assurance.
Affordable flow cytometry
Combined use of CD4 and CD45 conjugated antibodies using Panleucogating
(PLG) methodology (eg. Beckman Coulter FlowCARE CD4 Reagent) has been
found to be workable and cost-effective.
Modified single platform volumetric flow cytometry
Cyflow from Partec
The system works on volumetric method, i.e. a known amount of blood with
a single antibody reagent. It can also be run on solar panels and car batteries,
and hence may be used in remote areas. The methodology is simple to carry
Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
19
out based on an SP method with single antibody reagent and ten-minute
incubation. The system showed good correlation with the CD4 T lymphocytes
counts obtained by conventional flow cytometry. However, an experienced
technician is required for accurate measurement. The CyFlow capital equipment
costs approximately US$ 20 000 and the cost per test is US$2.
Guava technology
A SP system that uses CD3 antibodies to measure T lymphocytes and CD4
antibodies to estimate absolute T-cells expressing CD4. The system showed
good correlation with conventional flow cytometry and is easy to operate. It
uses smaller blood volume. It requires very minimal infrastructure facilities and
it is easy to train technologists to perform the test. The cost per test is around
US$ 2. However, the equipment costs about US$ 26 000.
The recent evaluation studies with these two modified flowcytometry
systems have shown a good correlation with the standard FCM.
Selection of alternative methodology for CD4 T
lymphocytes count
The following specifications should be considered for the selection of the better
technology for CD4 T lymphocytes count:
•
The equipment should be simple to operate, easy to maintain and
require minimal training.
•
Methodology needs minimal infrastructure laboratory facilities.
•
Methodology should have the internal QC procedures.
•
Test kits should be cost-effective and available anytime.
•
Easy access to the technical specialist/service engineer support.
•
Supplying company should be in a position to supply the critical parts
of the equipment with short turnaround time.
For HIV/ART monitoring, all CD4 T lymphocytes estimation for the same
patient must be done using the same technology and same laboratory to
ensure comparability.
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Guidelines for HIV Diagnosis and Monitoring of Antiretroviral Therapy
Utility of CD4 T lymphocytes count in monitoring
According to the WHO recommendation, HIV-infected adults should start ARV
therapy when infection has been confirmed and one of the following conditions
is present (Table 3.1):
Table 3.1: WHO recommendations for initiating ARV therapy in
HIV-infected adults and adolescents according to
CD4 T lymphocytes counts and total lymphocyte counts*
WHO Stage
CD4 T lymphocytes counts
Total lymphocyte counts
IV
Irrespective
Irrespective
III